Anti-Mel18 抗体 (ab5267)

Thank you for your enquiry. I have consulted the EXPASY databases and I retrieved the following information: Mel18 - Tissue specificity: Detected in all tissues examined with high expression found in placenta, lung and kidney and low expression, in liver, pancreas and skeletal muscle. Ring1B - No tissue specificity information. The recommended positive control for Mel18 is human lung lysate. For Ring1B we do not have a recommended positive control. However, a reviewer has successfully used this antiserum in western blotting using mouse cultured cells (3T3) and mouse brain. This may be a useful alternative to HeLa cells as I cannot guarantee that these cells express Ring1B. I have obtained the following details of how the positive control to how Mel18 was prepared and run by western blotting. Western Blot: Approx 40 kDa band observed in Human Lung extracts (predicted MW of 40kDa according to NP_009075). Recommended for use at 1-2µg/ml. Tissue Lysis. Tissue chunks were weighed and cut into approx 1mm cubes using a razor blade. The tissue was transferred to a handheld homogenizer and 3 ml of ice-cold RIPA buffer was added per 1g of tissue. The tissue was gently homogenised over 20 minutes on ice. The resulting lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS). SDS PAGE. Samples were run at 200V constant on a 12% acrylamide SDS-PAGE mini gel - using Biorad Mini-Protean 3 kit and protocols. Before loading samples had 5% (v/v) 2-ME added and were boiled for 3 minutes. Transfer. We used a Biorad Mini Trans-Blot, constant 100 V for 1 hour. Transfer Buffer was 20 mM Tris pH 8.0, 150 mM Glycine, 10% Methanol. We transferred to Millipore PVDF membrane and stained with Ponceau Red to evaluate the transfer. Staining. The membrane was blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05% Tween-20) for 1 hr at room temperature with agitation. Primary antibody was incubated for 1 hr at room temperature with agitation. We used [a competitor's] secondary 1:3000) for 1 hr at room temperature with agitation. We washed with TBST three times after primary and secondary antibody, each wash lasting for 5-10 mins. ECL-plus (Amersham) was used rather than ECL, which is considerably more sensitive. Final detection was on autoradiography film. I hope this information helps. Please do not hesitate to contact me should you require further assistance.